Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1,100 conditions) and 62 mouse studies (~2.4 million data points collected under 1,085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and reverse transcriptase polymerase chain reaction (RT-PCR) validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g., POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g., threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g., leukemia inhibitory factor (LIF)-activated JAK/STAT in mouse; NODAL/ACTIVIN-A-activated fibroblast growth factor in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation.

Germ cell tumors (GCTs) are unique in that they exhibit diverse biological characteristics and pathological features. Although several in vivo GCT models are available, studies on GCTs are hampered because in vivo development of GCTs is time consuming and prevents a detailed molecular analysis of the transformation process. Here we developed a novel strategy to transform mouse testis cells in vitro. Lentivirus-mediated transfection of dominant negative Trp53, Myc, and activated Hras1 into a CD9-expressing testis cells caused tumorigenic conversion in vitro. Although these cells resembled embryonic stem (ES) cells, they were aneuploid and lacked Nanog expression, which is involved in the maintenance of the undifferentiated state in ES cells. Euploid ES-like cells were produced by transfecting the Yamanaka factors (Pou5f1, Myc, Klf4, and Sox2) into the same cell population. Although these cells expressed Nanog, they were distinct from ES cells in that they expressed CD44, a cancer stem cell antigen. Both treatments induced similar changes in the DNA methylation patterns in differentially methylated regions of imprinted genes. Moreover, despite the differences in their phenotype and karyotype, both cell types similarly produced mixed GCTs on transplantation, which were composed of teratomas, seminomas, and embryonal carcinomas. Thus, in vitro testis cell transformation facilitates an analysis of the GCT formation process, and our results also suggest the close similarity between GCT formation and reprogramming.

The class I region of the human leukocyte antigen (HLA) complex located on chromosome 6p21.3 is gene dense. To define the gene content of the class I region, we are constructing genomic DNA feature maps. Here we report mapping of the POU5F1 and TCF19 genes to an approximately 0.2-Mb region between the HLA-C and the S genes. Localization of these genes was facilitated by subcloning genomic DNA fragments from the 0.2-Mb region into a transposon gamma delta-based vector, selecting transposon-mediated deletions in vivo in Escherichia coli, and sequencing a nested subset chosen for their uniform distribution of deletion endpoints. The POU5F1 and TCF19 genes are located approximately 130 kb telomeric of the HLA-C locus, approximately 0.6 kb apart from each other. Complete sequencing of a 5.5-kb EcoRI fragment containing the TCF19 gene revealed that it is composed of three exons, bounded by consensus splice signals. These experiments illustrate that the transposon-based nested deletion sequencing method provides an easy and efficient approach to feature mapping genomic fragments and to high-resolution analysis of selected subportions.

BACKGROUND

Mesenchymal stromal cells (MSC) can be obtained from potentially any tissue from the human body, but cells purified from different sources are undoubtedly different, and for each medical application, the MSC with the best regenerative potential should be chosen.

RESULTS

Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose tissue-derived mesenchymal stromal cells (AT-MSC) and Wharton's Jelly-derived mesenchymal stromal cells (WJ-MSC) were isolated from human tissues and were cultured under differentiation media supplemented with fetal bovine serum. We quantified the expression of stem cell and adipocyte genetic markers using quantitative real time PCR, as well as the secretion of cytokines, extracellular matrix components and growth factors using Luminex and ELISA. All three MSC differentiated into adipogenic cells. AT-MSC showed the highest shift in ADIPOQ, CEBPA and PPARG mRNA expression. BM-MSC kept high expression levels of stem-cell markers SOX2 and POU5F1. WJ-MSC showed the lowest increase in mRNA expression when cells were induced to differentiate into adipocytes. Regarding protein secretion, adipocyte-like cells generated from WJ-MSC secreted the highest chemokine levels. AT-MSC-derived adipocyte-like cells secreted the lowest cytokine amounts and the highest quantity of collagen types I and III. Adipocyte-like cells obtained from BM-MSC secreted high amounts of most angiogenic factors, growth factors TGF-β1 and TGF-β2, collagens type II and IV, heparan sulfate, laminin and aggrecan.

CONCLUSIONS

Mesenchymal stromal cells purified from different tissues have a different behavior when induced to differentiate into adipocyte-like cells.

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles - they were either methylated in pluripotent or non-pluripotent cells. The difference between these two β-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

Normal development depends on the precise sequence of changes in the configuration of chromatin; these are primarily related to specific biochemical modifications such as acetylation or methylation of histones and DNA methylation. While the role of DNA methylation during preimplantation development has been studied extensively, little is known about histone modifications related to early embryonic development. Here, we investigated gene-specific histone modifications in in vitro produced bovine blastocysts. Selected genes thought to be critical for bovine preimplantation development were examined and included POU5F1 (OCT4), NANOG, INFT, GAPDH, SLC2A3 and IGF1. We used chromatin immunoprecipitation from pools of bovine blastocysts to unravel several modifications of histone H3 in relation to mRNA expression profiles. We focused on the two cell compartments of the blastocyst, the inner cell mass (ICM) and the trophectoderm (TE). We show that gene expression patterns in the ICM and TE of the bovine blastocyst are consistent with histone modification patterns on the promoter of the corresponding genes. The data show a complex epigenetic pattern of promoter occupancy by transcriptionally permissive and repressive H3 modifications. These results pave the way to in-depth epigenetic studies of preimplantation embryos that are crucial to gain a better understanding of the epigenetic changes frequently observed after use of assisted reproductive technologies.

Although the establishment of putative porcine embryonic stem cells (ESCs) has been reported, such cell lines quickly lose their self-renewal ability, as they easily differentiate or become extinct after only a limited number of passages in culture. ESC-like cells exhibiting self-renewal rather than pluripotency are considered to be a valuable resource in applications such as drug screening and toxicology testing in humans, livestock and veterinary medicine. Here, we report the generation of unique cell lines established from the inner cell mass (ICM) of porcine embryos by using inhibitors of glycogen synthase kinase 3β and mitogen-activated protein kinase kinase 1. These ICM-derived cell lines were initially cultured and passaged in conventional ES medium for human ESCs and showed porcine ESC-like morphology with alkaline phosphatase (AP) activity. After transfer to culture in ES medium containing inhibitors, the morphology of the colonies was dramatically changed, i.e., they were closely packed smooth-edged colonies with close cell-cell boundaries and showed the expression of undifferentiated markers including OCT4 (POU5F1) and NANOG. Notably, the self-renewal capacity and morphology of the cells were LIF-dependent, consistent with the expression of LIF receptors and phosphorylation of signal transducer and activator of transcription 3. To date, our established cell lines have been cultured continuously for over 100 passages without any overt morphological changes. Thus, the established cell lines reported here provide a new ESC-like cell culture system for use not only in the fields of veterinary medicine and livestock but also human medical research, since porcine physiology closely resembles that of humans.

The homeodomain transcription factor, NANOG, along with OCT3/4 (POU5F1) and SOX2, is part of the core set of transcription factors that maintain embryonic stem cell self-renewal and pluripotency. Expression of NANOG has been detected in fetal germ cells and in gonadal germ cell tumors. To assess the diagnostic utility of NANOG in central nervous system (CNS) germ cell tumors, we analyzed its expression by immunohistochemistry in a series of 12 CNS germinomas and compared its expression with other stem cell markers. Strong nuclear expression of NANOG was demonstrated in >90% of the tumor cells in all cases. In contrast, expression of OCT3/4 and placental alkaline phosphatase was inconsistent and SOX2 was expressed in only rare cells. NANOG was not detected in tumor types frequently considered in the differential diagnosis of CNS germinoma: pineoblastoma, primitive neuroectodermal tumors, medulloblastoma, lymphoma, pituitary adenoma, atypical teratoid/rhabdoid tumor, Langerhans cell histiocytosis, and gliomas. These findings demonstrate that NANOG is a sensitive and specific marker of CNS germinoma. Compared with other currently used markers, NANOG may have superior diagnostic characteristics and can facilitate identification of germinomas in minute surgical biopsies commonly obtained from these tumors. These findings also suggest a potential biologic role for NANOG in maintenance of CNS germinoma.

BACKGROUND

Circular chromosome conformation capture, when coupled with next-generation sequencing (4C-Seq), can be used to identify genome-wide interaction of a given locus (a "bait" sequence) with all of its interacting partners. Conventional 4C approaches used restriction enzyme digestion to fragment chromatin, and recently sonication approach was also applied for this purpose. However, bioinformatics pipelines for analyzing sonication-based 4C-Seq data are not well developed. In addition, data consistency as well as similarity between the two methods has not been explored previously. Here we present a comparative analysis of 4C-Seq data generated by both methods, using an enhancer element of Pou5f1 gene in mouse embryonic stem (ES) cells.

RESULTS

From biological replicates, we found good correlation (r>0.6) for inter-chromosomal interactions identified in either enzyme or sonication method. Compared to enzyme approach, sonication method generated less distal intra-chromosomal interactions, possibly due to the difference in chromatin fragmentation. From all mapped interactions, we further applied statistical models to identify enriched interacting regions. Interestingly, data generated from the two methods showed 30% overlap of the reproducible interacting regions. The interacting sites in the reproducible regions from both methods are similarly enriched with active histone marks. In addition, the interacting sites identified from sonication-based data are enriched with ChIP-Seq signals of transcription factors Oct4, Klf4, Esrrb, Tcfcp2i1, and Zfx that are critical for reprogramming and pluripotency.

CONCLUSIONS

Both enzyme-based and sonication-based 4C-Seq methods are valuable tools to explore long-range chromosomal interactions. Due to the nature of sonication-based method, correlation analysis of the 4C interactions with transcription factor binding should be more straightforward.

Cancer stem-like cells (CSCs), a small population of pluripotent cells residing within heterogeneous tumor mass, remain highly resistant to various chemotherapies as compared to the differentiated cancer cells. It is being postulated that CSCs possess unique molecular mechanisms, such as autophagic homeostasis, that allow CSCs to withstand the therapeutic assaults. Here we demonstrate that HDAC6 inhibition differentially modulates macroautophagy/autophagy in CSCs as compared to that of differentiated cancer cells. Using human and murine CSC models and differentiated cells, we show that the inhibition or knockdown (KD) of HDAC6 decreases CSC pluripotency by downregulating major pluripotency factors POU5F1, NANOG and SOX2. This decreased HDAC6 expression increases ACTB, TUBB3 and CSN2 expression and promotes differentiation in CSCs in an apoptosis-independent manner. Mechanistically, HDAC6 KD in CSCs decreases pluripotency by promoting autophagy, whereas the inhibition of pluripotency via retinoic acid treatment, POU5F1 or autophagy-related gene (ATG7 and ATG12) KD in CSCs decreases HDAC6 expression and promotes differentiation. Interestingly, HDAC6 KD-mediated CSC growth inhibition is further enhanced in the presence of autophagy inducers Tat-Beclin 1 peptide and rapamycin. In contrast to the results observed in CSCs, HDAC6 KD in differentiated breast cancer cells downregulates autophagy and increases apoptosis. Furthermore, the autophagy regulator p-MTOR, upstream negative regulators of p-MTOR (TSC1 and TSC2) and downstream effectors of p-MTOR (p-RPS6KB and p-EIF4EBP1) are differentially regulated in CSCs versus differentiated cancer cells following HDAC6 KD. Overall these data identify the differential regulation of autophagy as a molecular link behind the differing chemo-susceptibility of CSCs and differentiated cancer cells.

Cancer stem cells play an important role in metastasis and the relapse of drug resistant cancers. Side-population (SP) cells are capable of effluxing Hoechst 33342 dye and are referred to as cancer stem cells. We investigated the effect of berberine on pancreatic cancer stem cells of PANC-1 and MIA PaCa-2. For both cell lines, the proportions of SP cells in the presence of berberine were investigated and compared to the proportions in the presence of gemcitabine, a standard pancreatic anti-cancer drug. The proportions of SP cells in the PANC-1 and MIA PaCa-2 cell lines were about 9 and <0.1%, respectively. After berberine and gemcitabine treatments, the SP cell proportion of PANC-1 decreased to 5.7 ± 2.0 and 6.8 ± 0.8%, respectively, which compares to the control proportion of (9.7 ± 1.7). After berberine and gemcitabine treatment of PANC-1, of the four stem cell-associated genes (SOX2, POU5F1, NANOG, and NOTCH1), all but NOTCH1 were down-regulated. Unfortunately, the effect of berberine and gemcitabine treatments on MIA PaCa-2 SP cells could not be clearly observed because SP cells represented only a very small proportion of MIA PaCa-2 cells. However, SOX2, POU5F1, and NANOG genes were shown to be effectively down-regulated in the MIA PaCa-2 cell line as a whole. Taken together, these results indicate that berberine is as effective at targeting pancreatic cancer cell lines as gemcitabine. Therefore, we believe that POU5F1, SOX2, and NANOG can serve as potential markers, and berberine may be an effective anti-cancer agent when targeting human pancreatic cancer cells and/or their cancer stem cells.

DNA methylation reprogramming plays important roles in mammalian embryogenesis. Mammalian somatic cell nuclear transfer (SCNT) embryos with reprogramming defects fail to develop. Thus, we compared DNA methylation reprogramming in preimplantation embryos from bovine SCNT and in vitro fertilization (IVF) and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos; however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, locus DNA methylation reprogramming showed three patterns in the SCNT group. First, some pluripotency genes (POU5F1 and NANOG) and repeated elements (satellite I and α-satellite) showed insufficient demethylation and hypermethylation in the SCNT group. Second, a differentially methylated region (DMR) of an imprint control region (ICR) in H19 exhibited excessive demethylation and hypomethylation. Third, some pluripotency genes (CDX2 and SOX2) were hypomethylated in both the IVF and SCNT groups. Additionally, VC improved the DNA methylation reprogramming of satellite I, α-satellite and H19 but not that of POU5F1 and NANOG in SCNT preimplantation embryos. These results indicate that DNA methylation reprogramming was aberrant and that VC influenced DNA methylation reprogramming in SCNT embryos in a locus-specific manner.

Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming.

Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.

The ovarian pool of follicle-enclosed oocytes is highly susceptible to elevated ambient temperature. It is not clear, however, whether the model of using heat shock in vitro simulates the effects of heat stress that animals experience in vivo. The current study examined the reliability of in vitro models, relative to in vivo models, for studying the effects of elevated temperature on the germinal vesicle (GV)-stage oocyte with emphasis on the expression of genes involve in maturation and early embryonic development. Cumulus oocyte complexes (COCs) were aspirated from ovaries arbitrarily collected at the slaughterhouse from multiparous Holstein cows. In the in vivo model, COCs were collected during the hot (May-September) and cold (December-April) seasons and then subjected to in vitro embryo production (IVP) at 38.5°C. In the in vitro model, COCs were collected during the cold season, pre-cultured with 75μM 3-isobutyl-1-methylxanthine (IBMX) for 16h at 38.5 or 41.2°C, and then subjected to IVP. For both models, the relative abundance of C-MOS, GDF9, GAPDH, and POU5F1 transcripts was examined in MII-stage oocytes by real-time PCR. Cleavage and blastocyst developmental rates were higher during the cold vs. hot season. IBMX pre-culture at 38.5°C successfully blocked resumption of meiosis without compromising further embryonic development, and the proportion of cleaved and developed embryos did not differ from the cold season. Exposure of GV-stage oocytes to 41.2°C reduced the proportion of cleaved oocytes developing to blastocysts relative to controls. The most prominent finding was that the relative abundance of the examined genes' transcripts was similarly reduced in heat-stressed oocytes from both models. The in vitro model was reliable and might be relevant for other environmental stressors as well.

Preimplantation embryo development is an important and unique period and is strictly controlled. This period includes a series of critical events that are regulated by multiple signal-transduction pathways, all of which are crucial in the establishment of a viable pregnancy. The p38 mitogen-activated protein kinase (MAPK) signalling pathway is one of these pathways, and inhibition of its activity during preimplantation development has a deleterious effect. The molecular mechanisms underlying the deleterious effects of p38 MAPK suppression in early embryo development remain unknown. To investigate of the effect of p38 MAPK inhibition on late preimplantation stages in detail, we cultured 2-cell stage embryos in the presence of SB203580 for 48 h and analysed the 8-cell, morula, and blastocyst stages. We determined that prolonged inhibition of the p38 MAPK altered the expression levels of Glut1 and Glut4, decreased glucose uptake during the 8-cell to blastocyst transition, changed the expression levels of transcripts which will be important to lineage commitment, including Oct4/Pou5f1, Nanog, Sox2, and Gata6, and increased cell death in 8-16 cell stage embryos onwards. Strikingly, while the expression levels of Nanog, Gata6 and Oct4/Pou5f1 mRNAs were significantly decreased, Sox2 mRNA was increased in SB203580-treated blastocysts. Taken together, our results provide important insight into the biological processes controlled by the p38 MAPK pathway and its critical role during preimplantation development.

Lung cancer is the leading cause of cancer-related human deaths. Exploration of the mechanisms underlying the metastasis of cancer stem-like cells (CSLCs) will open new avenues in lung cancer diagnosis and therapy. Here, we demonstrated that CSLCs-derived from lung adenocarcinoma (LAC) cells displayed highly invasive and migratory capabilities via expressing high levels of POU5F1 and MMP-2. We found that POU5F1 directly regulated MMP-2 transcription via interaction with the promoter of MMP-2. POU5F1 knockdown in LACSLCs reduced MMP-2 protein abundance, leading to inhibition of the cell invasion, migration and tumorigenesis potentials of LAC cells. Clinically, aberrantly high expressions of POU5F1 and MMP-2 were inversely correlated with the survival of LAC patients, and the double-positive POU5F1 and MMP-2 showed the worst prediction for the patient's poor survival. These results indicate that POU5F1 can bind to the MMP-2 promoter for the degradation of surrounding extracellular matrix, and therefore promote invasive and migratory capabilities of LACSLCs. Moreover, our data implicate that the pathological detection of the double-positive expressions for POU5F1 and MMP-2 will be useful as diagnostic and prognostic biomarkers in LAC to advance anti-metastasis therapy.

POU5F1 is a transcription factor and master regulator of cell pluripotency with indispensable roles in early embryo development and cell lineage specification. The role of embryonic POU5F1 in blastocyst formation and cell lineage specification differs between mammalian species but remains completely unknown in cattle. The CRISPR/Cas9 system was utilized for targeted disruption of the POU5F1 gene by direct injection into zygotes. Disruption of the bovine POU5F1 locus prevented blastocyst formation and was associated with embryonic arrest at the morula stage. POU5F1 knockout morulas developed at a similar rate as control embryos and presented a similar number of blastomeres by day 5 of development. Initiation of SOX2 expression by day 5 of development was not affected by lack of POU5F1. On the other hand, CDX2 expression was aberrant in embryos lacking POU5F1. Notably, the phenotype observed in bovine POU5F1 knockout embryos reveals conserved functions associated with loss of human embryonic POU5F1 that differ from Pou5f1- null mice. The similarity observed in transcriptional regulation of early embryo development between cattle and humans combined with highly efficient gene editing techniques make the bovine a valuable model for human embryo biology with expanded applications in agriculture and assisted reproductive technologies.

In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.

Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro.

BACKGROUND

Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries.

OBJECTIVE

Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue.

METHODS

We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis.

RESULTS

All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1α1], p = 0.016) and osteogenic-related genes (Runt-related transcription factor 2 [RUNX-2], p = 0.029; osteoclacin [OCN], p = 0.032; phosphate-regulating neutral endopeptidase, X-linked [PHEX], p = 0.0290, and POU domain class 5 transcription factor [POU5F], p = 0.016) and proteins (Noggin [NOG], p = 0.04, OCN, p = 0.02, RUNX- 2, p = 0.04, and substance P-1 [SP-1], p = 0.01) in the injured soft tissue, normalized to the contralateral limb and/or sham-treated naïve rats, increased on Days 3 to 14 postinjury. By 14 days, foci of hypertrophic chondrocytes, hyaline cartilage, and woven bone were present in the soft tissue surrounding the amputation site.

CONCLUSIONS

We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1α1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury.

CONCLUSIONS

The ability to correlate molecular events with histologic and morphologic changes will assist researchers and clinicians to understand HO and hence formulate therapeutic interventions.

We examined the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) for the first time. We evaluated the effects of pGM-CSF on SCNT-derived blastocyst formation and investigated gene expression. A total of 522 cloned embryos in 6 replicates were treated with 10 ng/ml pGM-CSF during in vitro culture (IVC). This treatment significantly (P<0.05) increased blastocyst formation and total cell number in blastocysts compared with the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no effect on cleavage rate. The numbers of cells in the inner cell mass and trophectoderm were significantly higher in the pGM-CSF treatment group (6.0 and 43.0, respectively) compared with the control (4.4 and 31.9, respectively). Treatment with 10 ng/ml pGM-CSF significantly increased POU5F1 and Cdx2 mRNA expression in blastocysts. In addition, Bcl-2, Dnmt1 and proliferating cell nuclear antigen (PCNA) mRNA expression were upregulated in blastocysts in the pGM-CSF supplemented group compared with the control. These results suggest that pGM-CSF improves the quality and developmental viability of porcine SCNT embryos by regulating transcription factor expression.

In mammalian ES cells, the transcription factors Klf4 and Klf2 contribute to maintenance of pluripotency and self-renewal and are regulated by Pou5f1/Oct4. In the early zebrafish embryo Pou5f1/Oct4 is necessary for expression of three Klf2/4 family members, klf2a, klf2b and klf17 (previously klf4b), similar to the regulation reported for mammalian ES cells. In this study, we analyzed blastula and gastrula stage Klf regulatory networks and their influence on zebrafish embryonic patterning. We show that Pou5f1 acts in combination with region-specific factors to activate klf2a, klf2b, and klf17 in the superficial cell layer of the embryo. In addition, Pou5f1 acts together with the BMP signaling pathway to activate and maintain expression of klf2a and klf2b in a ventral ectodermal domain. We used microarray expression profiles of klf2a, klf2b and klf17 knockdown and overexpression embryos to identify Klf target genes, which reveals that Klfs participate in specification of the extraembryonic enveloping layer (EVL). We discuss mechanistic implications of simultaneous activation of transcriptional targets by ubiquitous, like Pou5f1, and region-specific inducers, emerging as a common regulatory motif in early development.

Induced pluripotent stem cells (iPSCs) are generated via the expression of the transcription factors OCT4 (also known as POU5F1), SOX2, KLF4 and cMYC (OSKM) in somatic cells. In contrast to murine naive iPSCs, conventional human iPSCs are in a more developmentally advanced state called primed pluripotency. Here, we report that human naive iPSCs (niPSCs) can be generated directly from fewer than 1,000 primary human somatic cells, without requiring stable genetic manipulation, via the delivery of modified messenger RNAs using microfluidics. Expression of the OSKM factors in combination with NANOG for 12 days generates niPSCs that are free of transgenes, karyotypically normal and display transcriptional, epigenetic and metabolic features indicative of the naive state. Importantly, niPSCs efficiently differentiate into all three germ layers. While niPSCs can be generated at low frequency under conventional conditions, our microfluidics approach enables the robust and cost-effective production of patient-specific niPSCs for regenerative medicine applications, including disease modelling and drug screening.